Stem cells are by definition present in all self-renewing tissues. These cells are believed to be long-lived, have a great potential for cell division and are ultimately responsible for the homeostasis of steady-state tissues. Stem cells rarely incorporate radioisotopes after single pulse labeling indicating that they are normally slow cycling. They can, however, be induced to enter the proliferative pool in response to certain growth stimuli. When stem cells undergo occasional cell division, they give rise to more rapidly proliferating "transient amplifying cells" ("TA") which incorporate a radiolabel such as tritiated thymidine (.sup.3 H-TdR) after a single exposure.
Stem cells possess many of the following properties: they are relatively undifferentiated, ultrastructurally and biochemically; they have a large proliferative potential and are responsible for the long term maintenance and regeneration of the tissue; they are normally "slow-cycling", presumably to conserve their proliferative potential and to minimize DNA errors that could occur during replication; they can be stimulated to proliferate in response to wounding and to certain growth stimuli; they are often located in close proximity to a population of rapidly proliferating cells corresponding to the transient amplifying cells ("TA") in the scheme of (1) stem cell to (2) TA cell to (3) terminally differentiated cell, and they are usually found in well protected, highly vascularized and innervated areas.
Positive identification of stem cells has been difficult because there are no known immunological or biochemical markers specific for epithelial stem cells. Since they are normally "slow-cycling", they cannot be labeled by single pulse administration of radioactive materials typically used to detect actively proliferating TA cells. It has been found that labeling of stem cells requires continuous labeling for a prolonged period. Once labeled, these slow-cycling cells retain isotope for an extended period of time. Such cells have been termed "label-retaining cells" or "LRCs".
Cotsarelis et al., J. Invest. Dermol. 92(3) (1989a) disclose a method to facilitate detection of LRCs based on the ability of slow-cycling cells to be recruited to proliferate in response to hyperplastic stimuli. Alzet.TM. osmotic minipumps were intraperitoneally implanted in adult SENCAR mice to deliver 20 .mu.Ci of tritiated thymidine (.sup.3 -HTdR) per day for 14 days. During this labeling period, 0.01% O-tetradecanoylphorbol 13-acetate (TPA) in petroleum (Pet) was applied topically once daily for 4 days to the right flank. The contralateral side was treated with Pet only. Animals were sacrificed during and after labeling. TPA and Pet treated skin was examined by light microscopy and tissue section autoradiography. It was found that TPA treatment caused marked epidermal and follicular hyperplasia, whereas Pet treated sites did not appear morphologically altered. Fourteen days of continuous .sup.3 -HTdR resulted in greater that 90% labeling of all nucleated epidermal and follicular epithelial cells in both TPA and Pet treated sites. After 4 weeks, only a small number of cells remained labeled (LRCs). These cells were detected with greater frequency in TPA- vs. Pet-treated epidermis. The most striking concentration of LRCs was found to occur in the follicular epithelium.
Using tritiated thymidine (.sup.3 H-TdR) labeling, a subpopulation of corneal epithelial basal cells located in the peripheral cornea in a region called the limbus, were identified by Cotsarelis et al., in Cell 57:201-209 (1989b). These cells are normally slow-cycling but can be stimulated to proliferate in response to wounding and to administration of TPA. The corneal epithelium appears to represent an exceptional situation. LRCs were detected in the basal layer of limbal epithelium. No such cells were detected in central corneal epithelium. It was found that limbal epithelium can be selectively stimulated to proliferate by introducing a wound 1-2 mm away in the central corneal epithelium. Preferential stimulation of limbal epithelial proliferation was also observed when TPA was topically applied to the anterior surface of the eye. It was therefore concluded that the limbal epithelium has a greater proliferative potential than central corneal epithelium.
Label-retaining cells were identified in mouse epidermis by continuously labeling with .sup.3 H-TdR using subcutaneous injections for seven days. This method labeled almost all epidermal cells. After chasing for four weeks, it was found that a subpopulation of epidermal basal cells maintained labeled-LRCs.
In other experiments, Alzet.TM. osmotic minipumps were implanted intraperitoneally in mice to deliver .sup.3 H-TdR for 14 days. Radioactive nuclei of over 95% of corneal epithelial cells and over 80% of limbal epithelial cells was observed. After a four week resting period, all of the radiolabeled cells disappeared from the cornea and few if any could be identified in limbal epithelium, suggesting that LRCs of the corneal-limbal epithelia must have an average cycling time much longer than 14 days and are therefore refractory to labeling under these experimental conditions.
To improve the chances of labeling these stem cells, they were recruited into a proliferative phase by wounding and application of TPA. These experiments showed the existence of a subpopulation of limbal basal cells that are normally slow-cycling but can be induced to proliferate and become labeled after appropriate stimulation.
Stem cells of various epithelia share a common set of features which are summarized in FIG. 7 of Cotsarelis et al.(1989b). The specific location and biological properties of corneal epithelial cells as well as the stem cells of a number of other epithelia including palmar (palm) epithelium, trunk epidermis, hair follicle, dorsal tongue epithelium, and intestinal epithelium are discussed. In FIG. 7(e) it is shown that in hair follicles, the heavily pigmented stem cells are located at the base, in close proximity with follicular papillae and associated vasculature.
In subsequent work, Cotsarelis et al. (1990) show that the hair follicle stem cells were, however, incorrectly identified, Cotsarelis et al. (1989b). In fact, label-retaining cells were found to exist exclusively in the midportion of the follicle at the arrector pili muscle attachment site termed the "bulge" area of the hair follicle (Cell 61:1329-1337).